β-Lactam antibiotic, including penicillins, cephalosporins, monobactams, and carbapenems induces a death of live cell by inhibition of cell-wall synthesis (Tomasz, 1979). But it is induced an emergence of bacteria having a resistance for the above β-lactam antibiotics due to a broad use of these antibiotics. Expression of β-lactamase is a general resistance mechanism of bacteria for β-lactam antibiotics, which these enzymes hydrolyze a lactam-ring of the above antibiotics. β-Lactamase is classified into four classes of A, B, C, and D according to homology of amino acid sequence (Ambler, 1980).
The β-lactamase-mediated resistance of pathogenic bacteria to antibiotics is a continuing threat to public health. Therefore, a third generation of cephalosporins was developed that could escape inactivation by β-lactamases. The new antibiotics such as cefotaxime and ceftazidime contain bulky oxyimino group at the C7 position of cephalosporin nucleus. After clinical use, however, novel β-lactamases that could inactivate even the oxyimino β-lactams appeared. For example, the chromosomal class C β-lactamase that hydrolyzes the above oxyimino β-lactams has been isolated from the Gram-negative bacteria, Enterobacter cloacae strain GC1 (Nukaga et al., 1995).
Clinically, class A and C β-lactamase are the most commonly encountered of the four classes. However, class C β-lactamases are more problematic than class A enzymes. Class C β-lactamases can confer resistance to cephamycins (cefoxitin and cefotetan), penicillins, cephalosporins and β-lactam/β-lactamase inhibitor combinations and are not significantly inhibited by clinically used β-lactamase inhibitor such as clavulanic acid. In contrast, Class A β-lactamases are not able to confer resistance to cephamycins and the enzymes are generally susceptible to inhibition by clavulanic acid.
Class C β-lactamases are typically synthesized by the Gram-negative bacteria and are mainly chromosomal. Recently, plasmid-encoded class C β-lactamases have been reported in several bacteria species (Lee et al., 2002). Plasmid-encoded class C β-lactamases pose more problems since they are transmissible to other bacterial species and are often expressed in a large amount (Marchese et al., 1998).
CMY-1 is the first plasmidic class C β-lactamase to be identified. CMY-10 is a variant of the above CMY-1 with a point mutation at position 346 from Asn to Ile. CMY-1 and CMY-10 display the characteristics of extended-spectrum β-lactamase (ESBLs) (Lee et al., 2003; Horii et al., 1993). The above CMY-10 enzyme is able to hydrolyze cefoxitin and cefotetan as well as penicillins, the third-generation cephalosporins, and monobactams (Lee et al., 2003; Bauernfeind et al., 1989). The high sequence identity between plasmidic β-lactamase and chromosomal lactamase clearly defines the origin of the above plasmidic enzymes. Namely, MIR-1, plasmidic β-lactamase, shows over 90% sequence identity to a chromosomal enzyme AmpC from Enterobacter cloacae. P99 is not ESBL, but is wild type of GC1. In the case of CMY-1 and CMY-10, however, the root is obscure since there is no closely related chromosomal class C enzyme.
Structural information on class C β-lactamase is very restricted. All available structures have been determined using chromosomal β-lactamase (Crichlow et al., 1999; Lobkovsky et al., 1993; Oefner et al., 1990; Usher et al., 1998). Thus, the structure of CMY-1 and CMY-10 will open new opportunities for structural comparison between chromosomal and plasmidic class C β-lactamases and for the design of new antibiotics that can escape hydrolysis by plasmidic class C ESBLs.
With considering the above described state, therefore, it is found the CMY-10 gene present in the plasmid isolated from Enterobacter aerogenes has been over-expressed in E. coli, followed purification and crystallization of CMY-10 according to the present invention. It is also obtained X-ray diffraction data for CMY-10 crystal and determined a three-dimensional structure of the CMY-10 molecule using the above data.